A method for generating high density arrays of microscopic tissue samples on glass slides has been described and widely adopted (Kononen et al., (1998); Bubendorf et al., (1999); Schraml et al., (1999); Bubendorf et al., (2001a); Bubendorf et al., (2001b); U.S. Pat. No. 6,103,518 to Leighton et al.). However, current methodology to display high density arrays of tissue samples on glass slides for analysis (e.g., microscopy, histochemistry, immunohistochemistry, fluorescent in situ hybridization, etc.) is limited to densities of less than 100 features/cm2. Valuable space is wasted on the array by the residual space between circular features. The current requirement of space in the array is dictated by the cylindrical cores generated by current technology of drilling cores in the tissue or in the medium (e.g., plastic, paraffin, etc.).
Multiple tissues placed on a single pathology slide have been reported by Battifora et al. (U.S. Pat. No. 4,820,504). Using the so-called “sausage technique,” numerous tissue chunks were wrapped together in a sheath in an identifiable but disorganized manner for immunohistochemical studies. The arrays of Battifora contained only on the order of a 100 or so tissue features per slide.
Kononen and colleagues have described parallel display of up to 1,000 small tissue or tumor samples of 0.6 mm in diameter on a single microscope slide (about 2 cm×5 cm available surface) that can be processed simultaneously for direct microscopy or scanning or microscopy or scanning afterimmunohistochemical staining or other staining procedures (e.g., regular tissue staining with hematoxylin+eosin, fluorescence-based in situ mRNA or DNA analysis, etc.). The method of Kononen et al. combines innovative placement of tissue cores of up to 10 mm in height into predrilled holes in a paraffin block to assemble a high density of sample cores lined up in parallel. Once a complete collection of tissue cores has been placed in the same tissue block, transverse sections are cut by a microtome, and the resulting 3-10 μm thick sections are transferred to glass slides and adhered or cross-linked to the coated glass. The resulting microarray of small tissue samples can be duplicated many times by cutting multiple sections.
However, the method of Kononen et al. suffers from a number of limitations. Sample density is limited due to needed space between samples to maintain the integrity of the paraffin holding block. Currently, the maximal limit is 1,000 samples per slide (˜100 tissue features/cm2). The height of the tissue block is limited due to limitations in the height of each core (0.5 to 1 cm). This height restriction limits the number of theoretical sections to approximately 13,000 sections when cut at 3 μm thickness. This method also promotes inefficient use of samples. Because the method is based on drilling cores out of the primary sample, e.g., a tumor specimen, there will be wasted sample material left between the drilling holes. Core drilling results in reduced quality of the remaining tissue sample, because the integrity of the original sample is broken by the core(s) that are removed (i.e., a “Swiss cheese” effect is produced).
The method of Kononen et al. requires a certain structural rigidity of the sample to work. Softer tissues or materials will be crushed when attempting to remove the core from the drill. Moreover, the method can be most readily used with paraffin-embedded tissue and less readily on frozen tissue or softer gelatinous tissues or material. Finally, although a core-drilling, semi-automated tissue arraying machine has recently been introduced (ATA-27 Automated Arrayer, Beecher Instruments), the core-based semi-automated technology is limited to handling only a low number of specimens at a time, currently a maximum of twenty-six specimens due to the labor-intensive transfer of individual samples to the block. This method is also not readily fully automated due to the manual drilling of cores and placement of individual cores one by one into the predrilled holes. This problem is in part due to the need for movement of the tissue core from sample to block at a central site, with the very limited possibility of gathering and storing cores (samples) before assembly.
The present invention overcomes many of these problems and increases the density of array elements that can be achieved. While the present method is described in detail for tissue arrays, the method has widespread applications as discussed below.